Mycoplasma pneumoniae / chlamydia isothermal amplification technology combined colloidal gold detection method
By using RPA-LAMP cascade amplification and dual-labeled colloidal gold probe technology, the problems of primer design complexity and aerosol contamination in the detection of Mycoplasma pneumoniae and Chlamydia pneumoniae have been solved, enabling rapid and accurate dual pathogen detection in primary healthcare settings. This technology is suitable for simple operation in primary healthcare settings.
Patent Information
- Application Number
- CN202511177566.6
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-08-21
- Publication Date
- 2025-11-07
AI Technical Summary
In existing technologies, rapid on-site detection of Mycoplasma pneumoniae and Chlamydia pneumoniae suffers from complex primer design, high non-specific amplification rate, aerosol contamination affecting the accuracy of results, time-consuming and costly multi-pathogen detection, and colloidal gold detection requires specialized equipment and skilled operators, making it difficult to meet the needs of primary healthcare for simple, rapid, and closed-tube testing.
The simultaneous detection of Mycoplasma pneumoniae and Chlamydia pneumoniae was achieved by using recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP) cascade amplification technologies, combined with dual-labeled colloidal gold probes and immunochromatographic test strips. The target genes were enriched by RPA pre-amplification and LAMP targeted amplification. The dUTP/UDG enzyme system was used to prevent contamination and the operation was carried out in a closed container.
It significantly reduces primer design complexity, prevents aerosol contamination, and enables high-sensitivity simultaneous detection of two pathogens within 25 minutes. It is suitable for operation by primary healthcare personnel, reduces costs, and improves detection accuracy and safety.
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of molecular diagnosis, in particular to a Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with colloidal gold detection method. BACKGROUND
[0002] Current on-site rapid detection of Mycoplasma pneumoniae and Chlamydia mainly relies on nucleic acid isothermal amplification technology combined with colloidal gold chromatography. In the traditional method, isothermal amplification needs to design multiple primer sets to match complex targets, and secondary structures are easily formed between primers, resulting in an increase in non-specific amplification rate. The amplification product is stable, and when the cover is opened for detection, it is easy to form aerosol pollution, affecting the accuracy of the results. When multiple pathogens are detected simultaneously, the difference in primer annealing temperature limits the efficiency of synchronous amplification. The colloidal gold detection link mainly uses single-pathogen detection mode, and double-pathogen detection needs to be operated in batches, which is time-consuming and costly.
[0003] Existing colloidal gold test strips rely on antigen-antibody reactions, and the preparation cycle of monoclonal antibodies is long, and the stability between batches is poor. Cross-reactions between probes in multiplex detection can easily reduce sensitivity, and cannot distinguish between active infection and antibody residue. Traditional methods require special equipment and skilled operators, and cannot meet the core needs of "simplicity, rapidity, and closed tube detection" in primary medical care. SUMMARY
[0004] This section aims to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification to avoid obscuring the purpose of this section, abstract and title, and such simplifications or omissions cannot be used to limit the scope of the present application.
[0005] In view of the problems in the prior art, the present application is proposed.
[0006] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art and provide a Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with colloidal gold detection method.
[0007] To solve the above technical problems, the present application provides the following technical scheme: a Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with colloidal gold detection method, comprising the following steps: Step S1: using recombinase polymerase amplification (RPA) to pre-amplify Mycoplasma pneumoniae P1 adhesion protein gene and Chlamydia ompA gene in the sample, the reaction temperature is 37℃±1℃, and the time is 5±1 minutes; Step S2: transferring the RPA product into a loop-mediated isothermal amplification (LAMP) system, using FIP / BIP primer sets for Mycoplasma pneumoniae and FIP primers for Chlamydia for targeted amplification, the reaction temperature is 40℃±2℃, and the time is 15±3 minutes; Step S3: mixing the LAMP product with double-labeled colloidal gold probe, synchronously detecting Mycoplasma pneumoniae and Chlamydia through the immunochromatographic test strip, and the chromatography time is 10±2 minutes; In step S1, the pre-amplification target is the genomic DNA of Mycoplasma pneumoniae P1 gene and Chlamydia ompA gene.
[0008] As a preferred scheme of the Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with colloidal gold detection method, the RPA reaction system comprises recombinant enzyme UvsX 30-50 nM and single-strand binding protein gp3 100-150 nM.
[0009] As a preferred scheme of the Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with colloidal gold detection method, in the pre-amplification of step S1, the Mycoplasma pneumoniae primer targets the intra-species conserved region of P1 adhesion protein gene, and the Chlamydia primer targets the intra-species conserved region of ompA gene, and the conserved region is determined by multiple sequence alignment.
[0010] As a preferred scheme of the Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with colloidal gold detection method, in the LAMP amplification system of step S2, 0.3-0.8 mM dUTP and 0.5-2 U / μL uracil DNA glycosylase (UDG) are added.
[0011] As a preferred scheme of the Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with colloidal gold detection method, the 5' end of the double-labeled colloidal gold probe is modified with biotin, and the 3' end is modified with a reporter molecule, wherein the reporter molecule of the Mycoplasma pneumoniae probe is fluorescein (FAM), and the reporter molecule of the Chlamydia probe is digoxin (Digoxigenin).
[0012] As a preferred scheme of the Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with colloidal gold detection method, the immunochromatographic test strip comprises two independent detection lines, a Mycoplasma pneumoniae detection line (T1) embedded with anti-FAM antibody, and a Chlamydia detection line (T2) embedded with anti-digoxin antibody.
[0013] As a preferred scheme of the Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with colloidal gold detection method, step S3 specifically comprises: incubating the LAMP product with the colloidal gold probe in the chromatography buffer at room temperature for 3-8 minutes, and then adding to the sample addition hole of the test strip.
[0014] As a preferred scheme of the Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with colloidal gold detection method, the sample is respiratory tract secretion extract liquid, which is prepared by treating with lysis buffer (containing 0.1%-0.5% Triton X-100 and protease K) at 37°C-95°C for 3-20 minutes.
[0015] As a preferred scheme of the Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with colloidal gold detection method, the method is completed in a closed container, and the amplification product does not need to be transferred by opening the cover.
[0016] Application of the Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with colloidal gold detection method in rapid diagnosis of respiratory tract pathogens.
[0017] The application has the following beneficial effects: the RPA-LAMP cascade amplification system separates pre-amplification and target amplification. The RPA preliminarily enriches the target gene fragment, so that the subsequent LAMP only needs a core primer set, and the complexity of primer design and the risk of non-specific amplification are significantly reduced; the dUTP / UDG enzyme system is introduced to degrade the pollution DNA containing uracil at room temperature, and the aerosol pollution is completely eliminated in combination with the closed tube operation. The whole process does not need to open the cover for pipetting, and the operation safety is greatly improved. The double-labeled probe design separates the "biotin-reporter molecule" function, the Mycoplasma pneumoniae probe is labeled with FAM, the Chlamydia probe is labeled with digoxin, and the independent double detection lines of the test strip are matched to realize synchronous detection and zero cross reaction. The detection time is shortened to 25 minutes, the sensitivity is 10 copies / μL, the basic personnel can independently complete the detection after simple training, and the precise diagnosis is promoted to the community medical scene. DETAILED DESCRIPTION
[0018] In order to make the above-mentioned objects, features and advantages of the present application more apparent and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the embodiments of the present application.
[0019] In the following description, a large number of specific details are set forth in order to provide a thorough understanding of the present application, but the present application can also be implemented in other ways different from the description, and those skilled in the art can make similar generalizations without departing from the concept of the present application, so the present application is not limited to the specific embodiments disclosed below.
[0020] Secondly, the "one embodiment" or "embodiment" referred to herein means that the specific features, structures or characteristics can be included in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor does it mean an embodiment that is separate or selectively excluded from other embodiments.
[0021] Embodiment 1 The embodiment provides a Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with a colloidal gold detection method.
[0022] Specifically, a single Mycoplasma pneumoniae closed tube detection process is as follows: Sample processing: Specifically, a throat swab of a patient is collected and placed in 1 mL of a nucleic acid release agent, preferably containing 0.1% Triton X-100, 20 mM Tris-HCl buffer and 0.5 mM EDTA.
[0023] As an implementation manner, the sample is lysed at 37°C for 5 minutes, centrifuged at 12,000 rpm for 2 minutes, and the supernatant is taken as a template. Preferably, a DNase inhibitor (such as EDTA 0.5-1 mM) is added to the lysis buffer to avoid degradation of the target gene by DNA enzyme in the sample.
[0024] RPA pre-amplification: Specifically, the reaction system (25 μL) contains: recombinase UvsX 40 nM, single-strand binding protein gp32 120 nM, dNTPs 0.4 mM, Mycoplasma pneumoniae primers (targeting the conservative region of P1 gene) 0.2 μM each; Further, the reaction condition is 37°C constant temperature for 5 minutes.
[0025] LAMP target amplification: Preferably, 10 μL of RPA product is transferred to the LAMP system: Bst DNA polymerase 8 U, Mycoplasma pneumoniae FIP / BIP primer set 1.6 μM each, UDG enzyme 1 U / μL; Closed tube chromatographic detection: As a specific implementation manner, 0.5 μM of colloidal gold probe is directly added to the reaction tube, mixed, and then added dropwise to the sample well of the test strip. After chromatography for 10 minutes, the judgment is as follows: T1 line (anti-FAM antibody) shows red color for positive.
[0026] Embodiment 2 The embodiment provides a Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with a colloidal gold detection method.
[0027] Specifically, a double-pathogen combined detection and cross-prevention design is as follows: Probe and test strip construction: Specifically, two double-labeled probes are designed: Mycoplasma pneumoniae probe: 5' end biotin-3' end FAM; Chlamydia probe: 5' end biotin-3' end digoxin.
[0028] Preferably, the nitrocellulose membrane of the test strip is set as follows: T1 line: embedding anti-FAM monoclonal antibody (2 mg / mL); T2 line: embedding anti-digoxin monoclonal antibody (2 mg / mL); C line: embedding streptavidin (quality control line).
[0029] As a specific embodiment, the selection criteria for the conserved region are: ① BLAST alignment of 100 clinical isolates with sequence consistency ≥ 90%; ② No cross-homologous region Synchronous detection operation: as an embodiment, the LAMP product is incubated with double probes in the chromatography buffer at room temperature for 5 minutes, and then added dropwise to the test strip. The result is interpreted: T1 line coloration → Mycoplasma pneumoniae positive; T2 line coloration → Chlamydia positive; Double line coloration → mixed infection.
[0030] It should be noted that the Chlamydia positive sample (n=30) was used to detect the T1 line of Mycoplasma pneumoniae, and the coloration negative rate was 100%, which confirmed that the probe-antibody pairing had no cross reaction.
[0031] Example 3 The present embodiment provides an isothermal amplification technology combined with colloidal gold detection method for Mycoplasma pneumoniae / Chlamydia.
[0032] Specifically, the pollution prevention system and basic application verification: dUTP / UDG pollution prevention mechanism: specifically, 0.5 mM dUTP and 1 U / μL UDG enzyme are added in the LAMP system: Before amplification: UDG enzyme degrades the pollution DNA containing dUTP in the environment; After amplification: the newly produced product carries dUTP label, which can be degraded by UDG enzyme in subsequent operation.
[0033] Closed tube process operation: preferably, the whole process is completed in a closed reaction tube: RPA pre-amplification → adding LAMP reagents by piercing the inner septum → adding probes by piercing the second septum after amplification → chromatography detection.
[0034] Further, 50 clinical samples were compared between open and closed tube operations: Open group: false positive rate 18.7% (aerosol pollution); Closed tube group: false positive rate 0%.
[0035] Basic application scenario: in community clinics, medical staff operate after 30 minutes of training: Detect 120 suspected respiratory infection samples; Double pathogen joint detection time 25±3 minutes; The result coincidence rate with PCR rechecking is 100%.
[0036] Further, RPA pre-amplification as a preliminary step, in a low-temperature environment at 37°C, quickly enriches target gene fragments, and simplifies the complex primer system required by traditional LAMP to specific primer sets for core regions. This "pre-enrichment-precision amplification" hierarchical strategy essentially decouples the nucleic acid amplification process into two specialized stages: RPA undertakes the broad-spectrum capture task, and LAMP performs high-fidelity amplification, thereby significantly reducing the difficulty of primer design while maintaining high sensitivity.
[0037] The introduction of the dUTP / UDG system, however, gives the detection process "self-cleaning" ability at the molecular level. The specific hydrolysis of UDG enzyme on DNA contaminated with dUTP sets up a recognizable "molecular tag" for the amplification product, allowing environmental contaminants to be targeted for removal at room temperature. This biochemical anti-pollution mechanism forms a redundant protection with the physical isolation of closed tube operation, completely solving the problem of aerosol pollution in field detection.
[0038] In the aspect of dual-pathogen joint detection, the design of probe-antibody system realizes the separation of "recognition-reporting" function. In the dual-labeled probe, the biotin end serves as a universal anchor point, and FAM and digoxin reporter molecules become pathogen-specific signal sources. The spatial isolation of anti-FAM and anti-digoxin antibodies on the test strip eliminates the risk of cross-reaction, which is essentially ensured by physical partitioning and chemical orthogonal mechanisms to ensure detection specificity. It is worth noting that this design breaks the dependence of colloidal gold detection on specific antibodies for pathogens, and the reporter molecule-antibody pairing can be pre-fabricated as a universal module. When new detection targets are added, only the probe reporter molecule end needs to be replaced, significantly reducing the cost of technology expansion.
[0039] From the perspective of grassroots application, the closed tube operation process restructures the working paradigm of molecular diagnosis. The high-risk opening and pipetting steps in traditional detection are replaced by "piercing fluid transfer" of pre-packaged reagents, simplifying the operation to "sample addition-incubation-reading" in three steps, making precise molecular diagnosis truly suitable for grassroots personnel for the first time. More profoundly, the 25-minute detection time makes it possible for outpatient and emergency departments to achieve "testing as soon as possible", promoting the transformation of respiratory disease diagnosis and treatment from empirical medication to targeted therapy. This balance between ease of use and professionalism is the long-awaited breakthrough for grassroots medical care.
[0040] This scheme constructs a reusable technology framework, the RPA-LAMP cascade module can be flexibly replaced by target primers, the dUTP / UDG anti-pollution system can be transplanted to other amplification platforms, and the orthogonal design of probe-antibody supports flexible expansion of detection throughput. This modular architecture lays a technical foundation for the development of respiratory multi-pathogen joint detection chips in the future, and also highlights the strategic value of upgrading from single technology innovation to diagnostic systems.
[0041] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application is described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced, without departing from the spirit and scope of the technical solutions of the present application, which should be covered in the scope of the claims of the present application.
Claims
1. A method for detecting Mycoplasma pneumoniae / Chlamydia by isothermal amplification technology combined with colloidal gold, characterized in that: The method comprises the following steps: Step S1: pre-amplifying the mycoplasma pneumoniae P1 adhesion protein gene and chlamydia ompA gene in the sample by using recombinase polymerase amplification (RPA), and the reaction temperature is 37℃±1℃, and the time is 5±1 minutes; Step S2: transferring the RPA product into a loop-mediated isothermal amplification (LAMP) system, and using the FIP / BIP primer set for mycoplasma pneumoniae and the FIP primer for chlamydia for targeted amplification, and the reaction temperature is 40℃±2℃, and the time is 15±3 minutes; Step S3: mixing the LAMP product with a double-labeled colloidal gold probe, and synchronously detecting mycoplasma pneumoniae and chlamydia by using an immunochromatographic test strip, and the chromatography time is 10±2 minutes; In step S1, the pre-amplification target is the genomic DNA of the mycoplasma pneumoniae P1 gene and the chlamydia ompA gene.
2. The method for detecting Mycoplasma pneumoniae / Chlamydia isothermal amplification technology combined with colloidal gold according to claim 1, characterized in that: The RPA reaction system comprises recombinase UvsX 30-50 nM and single-strand binding protein gp3 100-150 nM.
3. The method according to claim 1, wherein the method is characterized by: In the pre-amplification of step S1, the mycoplasma pneumoniae primer targets the intraspecific conserved region of the P1 adhesion protein gene, and the chlamydia primer targets the intraspecific conserved region of the ompA gene, and the conserved region is determined by multiple sequence alignment.
4. The method for detecting Mycoplasma pneumoniae and Chlamydia isothermal amplification technology combined with colloidal gold according to claim 1, characterized in that: In the LAMP amplification system of step S2, 0.3-0.8 mM dUTP and 0.5-2 U / μL uracil DNA glycosylase (UDG) are added.
5. The method for detecting Mycoplasma pneumoniae and Chlamydia isothermal amplification technology combined with colloidal gold according to claim 1, characterized in that: The 5' end of the double-labeled colloidal gold probe is modified with biotin, and the 3' end is modified with a reporter molecule, wherein: The reporter molecule of the mycoplasma pneumoniae probe is fluorescein (FAM); The reporter molecule of the chlamydia probe is digoxigenin (Digoxigenin).
6. The method for detecting Mycoplasma pneumoniae and Chlamydia isothermal amplification technology combined with colloidal gold according to claim 1, characterized in that: The immunochromatographic test strip comprises two independent detection lines: the mycoplasma pneumoniae detection line (T1) is embedded with anti-FAM antibody; and the chlamydia detection line (T2) is embedded with anti-digoxin antibody.
7. The method for detecting Mycoplasma pneumoniae / Chlamydia pneumoniae using isothermal amplification technology combined with colloidal gold as described in claim 1, characterized in that: Step S3 specifically comprises: incubating the LAMP product with the colloidal gold probe in a chromatography buffer at room temperature for 3-8 minutes, and then adding it to the sample well of the test strip.
8. The method for detecting Mycoplasma pneumoniae / Chlamydia pneumoniae using isothermal amplification technology combined with colloidal gold as described in claim 1, characterized in that: The sample is respiratory tract secretion extract liquid, which is prepared by treating with a lysis buffer at 37℃-95℃ for 3-20 minutes.
9. The method for detecting Mycoplasma pneumoniae / Chlamydia pneumoniae using isothermal amplification technology combined with colloidal gold as described in claim 1, characterized in that: The method is completed in a closed container, and the amplification product does not need to be transferred by opening the cover.
10. The application of the mycoplasma pneumoniae / chlamydia isothermal amplification technology combined with colloidal gold detection method in the rapid diagnosis of respiratory tract pathogens according to any one of claims 1-9.